ORIGINAL RESEARCH

published: 14 July 2022

doi: 10.3389/fendo.2022.935905

Comparing Clinical and Genetic

Characteristics of De Novo and
Inherited COL1A1/COL1A2 Variants
in a Large Chinese Cohort of
Osteogenesis Imperfecta
Yazhao Mei , Hao Zhang * and Zhenlin Zhang *

Shanghai Clinical Research Center of Bone Disease, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong
University Affiliated Sixth People’s Hospital, Shanghai, China

Purpose: Nearly 85%-90% of osteogenesis imperfecta (OI) cases are caused by

autosome dominant mutations of COL1A1 and COL1A2 genes, of which de novo
mutations cover a large proportion, whereas their characteristics remain to be
Edited by: elucidated. This study aims to compare the differences in clinical and genetic
Giacomina Brunetti,
characteristics of de novo and inherited COL1A1/COL1A2 mutations of OI, assess the
University of Bari Aldo Moro, Italy
average paternal and maternal age at conception in de novo mutations, and research the
Reviewed by:
Carla Caffarelli, rate of nonpenetrance in inherited mutations.
University of Siena, Italy
Vijaya Kumar Pidugu,
Materials and Methods: A retrospective comparison between de novo and inherited
National Cancer Institute (NIH), mutations was performed among 135 OI probands with COL1A1/COL1A2 mutations.
United States Mutational analyses of all probands and their family members were completed by Sanger
*Correspondence: sequencing. A new clinical scoring system was developed to assess the clinical severity of
Zhenlin Zhang
zhangzl@sjtu.edu.cn OI quantitatively.
Hao Zhang
Results: A total of 51 probands (37.78%) with de novo mutations and 84 probands
zhanghaozcl@163.com
(62.22%) with inherited mutations were grouped by the results of the parental gene
Specialty section: verification. The proportion of clinical type III (P<0.001) and clinical scores (P<0.001) were
This article was submitted to
significantly higher in de novo mutations. Missense mutations covered a slightly higher
Bone Research,
a section of the journal proportion of de novo COL1A1 mutations (46.34%) compared with inherited COL1A1
Frontiers in Endocrinology mutations (33.33%), however, lacking a significant difference (P=0.1923). The mean BMD
Received: 04 May 2022 Z/T-score at the lumbar spine in de novo mutations was -2.3 ± 1.5, lower than inherited
Accepted: 17 June 2022
Published: 14 July 2022
mutations (-1.7 ± 1.8), but lacking statistical significance (P=0.0742). There was no
Citation:
significant difference between the two groups in OI-related phenotypes (like fracture
Mei Y, Zhang H and Zhang Z (2022) frequency, blue sclera, and hearing loss) and biochemical indexes. In de novo mutations,
Comparing Clinical and Genetic
the average paternal and maternal age at conception was 29.2 (P<0.05) and 26.8
Characteristics of De Novo and
Inherited COL1A1/COL1A2 (P<0.0001), respectively, which were significantly younger than the average gestational
Variants in a Large Chinese age of the population. Additionally, 98.04% of pedigrees (50/51) with de novo mutations
Cohort of Osteogenesis Imperfecta.
Front. Endocrinol. 13:935905.
were spontaneous conception. The rate of nonpenetrance of parents with pathogenic
doi: 10.3389/fendo.2022.935905 variants in the inherited mutation group was 25.64% (20/78).

Frontiers in Endocrinology | www.frontiersin.org 1 July 2022 | Volume 13 | Article 935905

Mei et al.
Conclusions: Our data revealed that the proportion of clinical type III and clinical scores were
significantly higher in de novo mutations than in inherited mutations, demonstrating that de
novo mutations are more damaging because they have not undergone purifying selection.
Keywords: osteogenesis imperfecta, COL1A1, COL1A2, de novo, inherited, clinical scoring system
INTRODUCTION
Osteogenesis imperfecta (OI) is a generalized connective tissue
disorder, apart from liability to slight trauma fractures, the
phenotypes usually extend to non-skeletal symptoms, like blue
sclera, dentinogenesis imperfecta, hearing loss, and ligamentous
laxity (1). Based on the clinical and radiographic manifestations,
Sillence et al. (2) in 1979 proposed the classic classification of OI:
type I-IV: type I is the mildest kind characterized by nondeforming
with persistently blue sclerae; type II is the perinatal lethal form;
type III is the severest among surviving patients and presents with
progressively deforming; type IV is characterized by white sclerae,
whose severity is between type I and type III.
Although an increasing number of genes have been identified
in OI involving different modes of inheritance (3), about 85%-
90% of OI cases are caused by autosome dominant mutations of
COL1A1 and COL1A2 genes. They encode the a1(I) and a2(I)
chains of type I collagen, which is the main component of the
extracellular matrix of bone and skin and is responsible for their
elastic properties (4). Mutations in COL1A1 and COL1A2 of
collagen-related OI can be divided into two groups: quantitative
defect (haploinsufficiency) with synthesizing about half the
amount of structurally normal type I collagen, primarily
resulting from nonsense, frameshift, and some splice-site
variants; another is the structural defect, in which over 80%
mutations are the results of glycine substitutions in the helical
domain (4). The majority of the type II-IV are the consequences
of a structural defect because it has more severe impacts on the
extracellular matrix than haploinsufficiency does.
Parental genetic characteristics will be passed on to the next
generation. At the same time, de novo mutations, a kind of novel
genetic change in a family, will occur during the formation of the
gametes or postzygotic in each of us (5, 6). Only mutations
present in the germ cells can be passed on to the next generation
(7). Distinct from inherited variants, de novo mutations haven’t
undergone purifying selection during the reproductive phase,
thus making them more harmful (8). Approximately 80% of all
de novo germline point mutations occur on the paternal allele
because oocytes experience only one additional round of DNA
replication during their evolution into a mature ovum, whereas
spermatogonia will encounter hundreds of DNA replication and
cell fission before becoming sperm cells. What’s more, it has been
demonstrated that most types of de novo mutations are much
more correlated with increased paternal age at birth (9–11). Rare
sporadic diseases in the population are associated with de novo
mutations (12). In OI, nearly half of the cases are derived from
de novo mutations (13–15).
Previous studies have elucidated the genotype-phenotype
correlation and mutation spectrum of collagen-related OI
Frontiers in Endocrinology | www.frontiersin.org
De Novo and Inherited Mutations
through large cohorts (15–18), which contributes to achieving
a more accurate and economical diagnosis of OI. However, in
addition to some reports of de novo mutation cases in OI (16, 19),
few large cohort studies have compared de novo with inherited
mutations in collagen-related OI (14). In this study, we enrolled
135 Chinese probands of OI with de novo or inherited COL1A1/
COL1A2 mutations in the department of Osteoporosis and Bone
Disease of Shanghai Jiao Tong University Affiliated Sixth
People’s Hospital, and then compared the gene mutation
spectrum and phenotypic characteristics to investigate
differences between de novo and inherited mutations among
OI with COL1A1/COL1A2 mutations.
MATERIALS AND METHODS
Subjects
We carried out a retrospective review of the electronic medical
record from January 2005 to January 2022 for all patients at the
department of Osteoporosis and Bone Disease of Shanghai Jiao
Tong University Affiliated Sixth People’s Hospital who were
clinically diagnosed as OI (Figure 1). Our inclusion criteria were
(1): probands were confirmed with COL1A1 or COL1A2
mutation; (2) pedigrees had undergone parental gene
verification. The Ethics Committee of Shanghai Jiao Tong
University Affiliated Sixth People’s Hospital approved the
study. Written informed consent was obtained from patients
or legal guardians of children younger than 18.
Clinical Evaluation
Clinical information, including age at diagnosis, gender, family
history, non-skeletal features (like blue sclera, dentinogenesis
imperfecta, and hearing loss), and medical history were collected.
As for de novo pedigrees, the parental age at conception, methods
of conception, and reproductive times were also collected.
Information on bone fractures was evaluated, including site,
time of initiation, and frequency (per year). The patient’s
height was adjusted to standard deviation scores (SDS) based
on reference data from the Chinese National Centers for Disease
Control and Prevention (20). Baseline bone mineral density
(BMD) at the lumbar spine (before bisphosphonate or
denosumab treatment) was measured by dual-energy X-ray
absorptiometry (DXA) (Lunar, Madison, WI, USA; Hologic,
Boston, MA, USA). The coefficient of variability (CV) values of
the BMD for the Lunar device at L1-L4 and femoral neck were
1.39% and 2.22%, respectively (21). The CV values of the BMD
for the Hologic device at L1-L4 and femoral neck were 0.9% and
0.8%, respectively (21). The results were transformed into age-
and sex-specific Z scores (T-scores for males over 50 years old
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Mei et al. De Novo and Inherited Mutations

FIGURE 1 | Flowchart of study cohort selection.

and postmenopausal females) by combing reference data (22–
24). In addition, X-ray radiography of the upper and lower
extremities and thoracolumbar vertebrae were examined. Then
based on the clinical and radiological characteristics, probands
were categorized as clinical type I, III, or IV according to the
Sillence classification (2). Type II OI was not included in this
study as it’s a lethal perinatal form.
Apart from the Sillence classification, we also proposed a new
clinical scoring system to assess the severity of OI quantitatively
by referring to the scoring criteria of the previous OI researches
(18, 25, 26). Clinical score = number of fractures (1-3) + fracture
frequency (per year) (1-3) + bowing of lower limbs (0-4) +
scoliosis (0-2) + height standard deviation score (SDS) (1-4) +
BMD Z/T-score (1-4). The specific contents are displayed in
Table 1. The higher the clinical score, the more severe the
clinical phenotype.
Serum levels of calcium (Ca), phosphate (P), and alkaline
phosphatase (ALP) were measured by the Hitachi 7600-020
automatic biochemistry analyzer. Bone formation markers
osteocalcin (OC), the bone resorption marker serum beta cross-
linked C-terminal telopeptide of type 1 collagen (b-CTX), intact
parathyroid hormone (PTH), and 25-hydroxyvitamin D
(25OHD) were measured using the Cobas 6000 auto-analyzer
(Roche Diagnostics, Mannheim, Switzerland).
COL1A1/COL1A2 Gene Mutation Analysis
via Sanger Sequencing
Genomic DNA was extracted from the peripheral blood of all
probands and their parents by standard techniques using a DNA
extraction kit (Lifefeng Biotech, Shanghai). Exons and exon-
intron boundaries of COL1A1 and COL1A2 genes were amplified
by polymerase chain reaction (PCR) (GenBank accession NO.
NC_000017.11 and NO. NC_000007.14). Primers were designed
by the Web-based Primer 3 software (https://bioinfo.ut.ee/
primer3-0.4.0/). Direct sequencing was performed using
BigDye Terminator Cycle Sequencing Ready Reaction Kit, v.
3.1 (Applied Biosystems, California, USA). The products were
analyzed with an ABI 3730 sequencer (Foster, CA, United

TABLE 1 | The proposed clinical scoring system for osteogenesis imperfecta patients.

Score Score Score Score

Number of fractures Number<10 1 10 ≤ Number < 30 2 Number ≥ 30 3

Fracture frequency (per year) Frequency ≤ 1 1 1 < Frequency < 3 2 Frequency ≥ 3 3
Bowing of lower limbs No 0 Milda 2 Severe 4
Scoliosis No 0 Milda 1 Severe 2
Height SDS SDS ≥ -2 1 -3 ≤ SDS < -2 2 -4 ≤ SDS < -3 3 SDS < -4 4
BMD Z/T-score Z/T ≥ -2 1 -3 ≤ Z/T < -2 2 -4 ≤ Z/T < -3 3 Z/T < -4 4
a
only radiographic findings and no obvious impact on life.
Total score = 20.

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Mei et al.
States). SNPs were identified using Polyphred (https://droog.gs.
washington.edu/polyphred/). Then, the parental gene
verification was performed in the probands’ parents and
siblings by Sanger sequencing. Results absent from the
Osteogenesis Imperfecta Variant Database (https://oi.gene.le.ac.
uk/) were regarded as novel mutations.
Statistical Analysis
Measurement data (including age at diagnosis, height SDS, BMD
Z/T-score, fracture frequency, and clinical score) were presented
as mean ± standard deviation, and abnormal distributions were
presented as medians (Q1, Q3). The differences between de novo
and inherited groups were analyzed as appropriate by Grouped
T-Test or Non-parametric Test. Enumeration data (like gender
and COL1A1/COL1A2) were expressed as numbers (%) or ratios,
and intergroup differences were evaluated with Chi-Square Test
or Continuity Adjustment Chi-Square Test as appropriate. The
statistical analyses were performed by SAS 8.2 (SAS Institute Inc.,
Cary, NC, USA). A two-sided P < 0.05 was considered
statistically significant.
RESULTS
Altogether, 135 probands from 135 unrelated and non-
consanguineous pedigrees, including 118 patients who had
been published in our previous studies (15, 27, 28), were
enrolled in the study, among which 51 probands (37.78%)
belonged to the de novo mutation group, and the remaining 84
probands (62.22%) were from the inherited mutation group. The
details of clinical characteristics of each proband, COL1A1 and
COL1A2 mutation loci identified in the present study and the
parental gene verification are listed in Supplementary
Tables 1–4.
Clinical Characteristics of Probands in
De novo and Inherited Mutations
The comparisons of clinical characteristics of probands between
de novo and inherited mutations were summarized in Table 2.
Among 51 de novo mutation cases, twenty-one (41.18%)
probands with de novo mutation had at least one unaffected
sibling, and one proband (P37, 1.96%) had an affected twin sister.
The mean height SDS in de novo mutations was -2.3 ± 3.0, lower
than inherited mutations (-1.4 ± 1.9), and the mean BMD Z/T-
score at the lumbar spine was -2.3 ± 1.5, also lower than inherited
mutations (-1.7 ± 1.8), however, both of them lacking
significance (P=0.0767 and 0.0742, respectively). Like inherited
mutations, blue sclera covered a substantial proportion of de
novo mutations (82.22%), while hearing loss was relatively
infrequent (2.22%). There were no significant differences in age
at diagnosis, gender, three phenotypes (including the blue sclera,
dentinogenesis imperfecta, and hearing loss), and fracture
frequency between de novo and inherited mutations. As for
biochemical indexes (Ca, P, ALP, b-CTX, OC, PTH, and
Frontiers in Endocrinology | www.frontiersin.org
De Novo and Inherited Mutations
25OHD), no statistical difference was identified, either. X-rays
radiography of the lower extremities of six de novo and two
inherited mutation probands with clinical type III were exhibited
in Figure 2. Severe lower limb deformities of these probands
could be seen, which resulted in limited movement in their daily
life. Although there was no significant difference in the
proportion of type I and type IV between de novo and
inherited mutations, the proportion of type III in the de novo
mutation group was significantly higher than that in the
inherited mutation group (P =0.0002).
A total of 20 probands’ fathers or mothers confirmed with
pathogenic variants among 84 hereditary cases exhibited no OI-
related phenotype or fracture history, that is, nonpenetrance
(listed in Supplementary Table 3). The available data of parents’
medical history was 78. Hence, the rate of nonpentrance in the
inherited mutation group was 25.64% (20/78).
Genetic Analysis of COL1A1/COL1A2 in
De Novo and Inherited Mutations
All pedigrees in our study had completed parental gene
verification to confirm the mutation mode. A total of 123
different collagen type I variants were identified in 135
probands, including 65 (52.85%) missense mutations, 20
(16.26%) nonsense mutations, 21 (17.07%) frameshift
mutations, and 17 (13.82%) splice mutations (Figures 2I, J).
Of the 123 mutations, two variants (c.2669G>C in COL1A1, and
c.3583T>C in COL1A2) absent in the Osteogenesis Imperfecta
Variant Database (https://www.le.ac.uk/genetics/collagen/index.
html) were novel mutations.
The mutation spectrum of this study comprised four kinds of
mutation types, including missense, nonsense, frameshift, and
splice mutation. The comparisons between de novo and inherited
mutation spectrum were summed up in Table 2. COL1A1 and
COL1A2 mutation cases covered a similar proportion in the de
novo and inherited groups (P=0.1134). Probands with COL1A1
de novo mutations were 41, containing 19 (46.34%) missense
mutations, and probands with COL1A1 inherited mutations were
57, including 19 (33.33%) missense mutations. The missense
mutation in the de novo COL1A1 mutation group occupied a
larger percentage than in the inherited COL1A1 mutation group,
but lacking significance (P=0.1923). All ten probands with
COL1A2 de novo mutations were missense mutations, and
92.59% of twenty-seven probands with COL1A2 inherited
mutations were missense mutations. No significant intergroup
difference in the proportion of missense mutation of COL1A2
was discovered (P>0.9999). Glycine substitutions covered no
significantly different proportion of missense mutations in de
novo and inherited COL1A1/COL1A2 mutations. Additionally,
p.Gly337Ser was seen four times in the COL1A2 inherited
mutation group. Intriguingly, mutations of p.Gly821Ser,
p.Arg697*, and p.Arg1026* in COL1A1, and p.Gly694Asp in
COL1A2 shared in de novo and inherited mutation groups
(Table 3). The concrete proportions of each mutation type in
COL1A1/COL1A2 for two groups were displayed in
Supplementary Figure 1.
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Mei et al. De Novo and Inherited Mutations

TABLE 2 | Clinical and genetic characteristics of de novo and inherited COL1A1/COL1A2 mutations of OI.

De novo (n=51) Inherited (n=84) P-

value
Number of available Mean ± SD/n (%)/ratio/median Number of available Mean ± SD/n
data (Q1, Q3) data (%)/ratio/median
(Q1, Q3)

Clinical characteristics
Age at diagnosis (years) 51 13.6 ± 10.3 84 14.5 ± 10.1 0.6221
Gender (male/female) 51 32/19 84 57/27 0.5434†
Height SDS 41 -2.3 ± 3.0 76 -1.4 ± 1.9 0.0767
BMD (g/cm2) 29 0.6 ± 0.2 64 0.7 ± 0.2 0.5173
BMD Z/T-score at the lumbar spine 33 -2.3 ± 1.5 64 -1.7 ± 1.8 0.0742
Fracture frequency (per year) 46 1.6 ± 1.0 76 1.4 ± 0.7 0.1212
Blue sclera 45 37 (82.22%) 77 60 (77.92%) 0.5702†
Dentinogenesis imperfecta 45 16 (35.56%) 77 24 (31.17%) 0.6185†
Hearing loss 45 3 (2.22%) 77 6 (7.79%) 0.8970‡
Ca (mmol/L) 19 2.5 ± 0.1 34 2.4 ± 0.1 0.1696
P (mmol/L) 19 1.4 ± 0.3 35 1.4 ± 0.3 0.9899
ALP (U/L) 19 200.0 (85.0, 289.0) 35 213.0 (103.0, 0.8753¶
277.0)
b-CTX (ng/L) 14 724.0 (347.5, 1286.0) 28 786.2 (428.1, 0.9685¶
1154.0)
OC (ng/mL) 13 57.2 (26.8, 180.1) 27 61.6 (40.2, 95.9) 0.7539¶
PTH (pg/mL) 19 31.3 (18.8, 45.2) 33 37.9 (27.0, 54.4) 0.0569¶
25OHD (ng/mL) 16 19.7 (12.0, 40.0) 32 22.0 (17.1, 30.3) 0.4038¶
Clinical type I 46 26(56.52%) 77 55 (71.43%) 0.0916†
Clinical type III 46 16 (34.78%) 77 6 (7.79%) 0.0002†
Clinical type IV 46 4 (8.70%) 77 16 (20.78%) 0.0789†
Genetic characteristics
COL1A1/COL1A2 51 41/10 84 57/27 0.1134†
Missense mutation of Total 41 19 (46.34%) 57 19 (33.33%) 0.1923†
COL1A1 Gly substitution 19 14 (73.68%) 19 14 (73.68%) >0.9999†
Missense mutation of Total 10 10 (100.00%) 27 25 (92.59%) >0.9999†
COL1A2 Gly substitution 10 9 (90.00%) 25 23 (92.00%) >0.9999†
Clinical score Total 32 9.1 ± 4.7 63 5.8 ± 2.0 0.0005
COL1A1 Total 26 8.2 ± 4.6 44 5.6 ± 1.8 0.0092
Structural 11 12.6 ± 4.0 13 5.8 ± 1.9 0.0002
mutation
COL1A2 Total 6 13.0 ± 3.2 19 6.2 ± 2.3 0.0020
† ‡ ¶
Data are shown as mean ± standard deviation, numbers (%), ratios, or median (Q1, Q3). Chi-Square Test, Continuity Adjustment Chi-Square Test, Non-parametric Test, otherwise
Grouped T-Test.
Bold P values indicate significance.
SDS, standard deviation scores; BMC, bone mineral content; BMD, bone mineral density; Ca, calcium; P, phosphate; ALP, alkaline phosphatase; b-CTX, beta cross-linked C-terminal
telopeptide of type 1 collagen; OC, osteocalcin; PTH, intact parathyroid hormone; 25OHD, 25-hydroxyvitamin D.

Genotype-Phenotype Correlations in
De Novo and Inherited Mutations
The frequency of clinical type in each mutation type in de novo
and inherited mutations was exhibited in Figure 3. Both in de
novo and inherited mutation groups, mutation types of
nonsense, frameshift, and splice mainly resulted in clinical type
I, and clinical type III and IV (especially type III) were primarily
correlated with missense mutations. Compared with inherited
mutations in COL1A1/COL1A2, probands with de novo
mutations had significantly higher clinical scores (P = 0.0005)
(Table 2), which denoted more severe phenotypes. We also
compared clinical scores about COL1A1(P = 0.0092), missense
mutation of COL1A1(P=0.0002), and COL1A2(P=0.0020)
between two groups, respectively, and their significant results
were consistent with the outcome mentioned above. At the same
time, we presented clinical scores of available probands on
different de novo or inherited COL1A1/COL1A2 mutation loci
with the help of bubble plots (Figure 3). From four different
bubble plots, we could find that: firstly, there was no noticeable
difference in the mutation site distribution for the COL1A1/
COL1A2 gene between de novo and inherited mutations;
secondly, the clinical score of missense mutation was higher
than the other three mutation effects in general; lastly, bubbles of
the de novo COL1A1/COL1A2 mutations distributed higher in
the axis than the inherited did, which means the clinical scores of
de novo mutations were higher and phenotypes were more
severe. Four mutation loci shared in de novo and inherited
mutation groups, mentioned above, had a consistent clinical
type and clinical score between the two groups, except for one
case with p.Arg1026* mutation (Table 3).
The Parental Age at Conception in
De Novo Mutations
In the de novo mutation group, the average paternal and maternal
age was 29.2 and 26.8, respectively. In the US, National Vital
Statistics System data revealed that the average paternal age at

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Mei et al. De Novo and Inherited Mutations

FIGURE 2 | X-ray radiography of the lower extremities of six de novo and two inherited mutation probands with clinical type III, and diagrams showing mutation loci
identified in this study. (A, B) X-ray radiography of the femurs of the probands (p68 and p56) with inherited mutations. (C–H) X-ray radiography of the lower
extremities of the probands (P9, P3, P49, P43, P13, and P26, respectively) with de novo mutations. (I) Mutation loci in COL1A1 gene identified in two groups. (J)
Mutation loci in COL1A2 gene identified in two groups.

birth was 30.9 in 2015 (29), which was older than the mean
paternal age at conception in our study (P = 0.0236). The average
maternal age at conception in China in 2017 was 29.7 (30), which
was also older than our research data (P <0.0001). Additionally,
except for one proband (P36) who was the outcome of medically
assisted reproduction, the remaining 50 pedigrees (98.04%) with
de novo mutations were spontaneous conception.
DISCUSSION
Previous studies had illustrated the proportion of de novo
mutations in OI with COL1A1/COL1A2 mutations: 41.03% in
South Korea (13), 56.16% in Estonia, Ukraine, and Vietnam (14),
and 54.6% in our previous report (15). The percentage of de novo
mutations in our study (37.78%) was significantly lower than our
last report (P = 0.0029), which may be due to we excluded cases
without performing parental gene verification. OI is a
phenotypically heterogeneous disease that familial cases with the
same mutation can present remarkably phenotypic diversity,
ranging from mild to severe (31, 32). In our study, the rate of
nonpenetrance of hereditary probands’ parents was up to 25.64%.
Therefore, some affected parents of hereditary probands exhibited
very mild or even without symptoms, which may result in missed
diagnosis and increasing the false-positive rate of de novo
mutations if not performing parental gene verification.
Low bone mass is always presented in patients with OI, and in
our study, the de novo mutation group exhibited a lower BMD Z/
T-score at the lumbar spine, however, not significantly lower
than the inherited group. Fractures of long bones and vertebral
compression are the most notable features of OI, while there was
no difference in the fracture frequency between de novo and
inherited mutation groups in our study. Skeletal deformities like
bowing of long bones and scoliosis are usually associated with
severe type III. Apart from congenital causes, recurrent fractures
of extremities can also result in deformities. As displayed in
Figure 2, lower limb deformities would seriously damage the
ability to move, and most of them had no alternative but to be in

TABLE 3 | Mutations shared in de novo and inherited mutation groups.

Gene Amino acid Mutation De novo Inherited

change effect
Frequency Proband Clinical Clinical Frequency Proband Clinical Clinical
ID type score ID type score

COL1A1 p.Gly821Ser Missense 1 P5 III 15 1 p5 III N/A

p.Arg697* Nonsense 1 P30 IV 4 1 p20 I 5
p.Arg1026* Nonsense 1 P19 I 4 2 p8 N/A N/A
p52 I 6
COL1A2 p.Gly694Asp Missense 1 P44 III 15 1 p68 III 12

N/A, not available.

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Mei et al. De Novo and Inherited Mutations

A B

C D

E F

FIGURE 3 | Genotype-phenotype correlations in de novo and inherited mutations. (A, B) Frequency of clinical type for each mutation type in De novo and Inherited.
(C–F) The bubble plots of the clinical score of available probands with de novo or inherited COL1A1/COL1A2 mutations. X-axis represented the mutation site on the
COL1A1/COL1A2 gene; Y-axis was the clinical score; the colors of the bubble represented different mutation effects (missense, nonsense, frameshift, and splice,
respectively); the size of the bubble indicated the mutation frequency of each mutation site in the de novo or inherited mutation group. The bubble was higher and
bigger; the phenotype was more severe, and the mutation site was more frequent.

a wheelchair for the rest of their lives. Most of our patients’ severe
lower limb deformities were accompanied by severe scoliosis.
Activity limitations and participation restrictions are linked to
lower quality of life (QOL) (33), and physical QOL is worse in
children with more severe OI than in mildly affected children. In
an aspect of non-skeletal manifestation, blue sclera was the most
prevalent feature of patients with OI (28), and consistent with the
previous studies (34, 35), dentinogenesis imperfecta covered a
higher proportion in type III and IV OI than type I in our study.
Biochemical indexes including bone turnover markers showed
no statistical differences in our study. Several papers have
revealed that there were no significant differences in levels of
the bone turnover markers between different OI clinical types or
different OI mutation types (36, 37), therefore, the relationship
between severity and bone turnover rates in OI may be
more complicated.
Generally, haploinsufficiency of COL1A1 results in the
mildest form of OI. In contrast, haploinsufficiency of COL1A2
results in no apparent OI-related phenotype, and homozygous
null mutations of COL1A2 bring about phenotypes of varying
severity (38). However, in our study, one case (p61, c.1557
+3A>G) with haploinsufficiency of COL1A2 mutation showed
severe phenotypes and was classified as type III. Most of the
structural defects are missense mutations, in which over 80% of
mutations are the results of glycine substitutions in the Gly-Xaa-
Yaa repeat (4). The most frequent cause of OI with COL1A2
mutation is the structural defect of type I collagen, especially
glycine substitutions in the helical domain (16, 39). Consistently,
both de novo and inherited COL1A2 mutations in our study were
predominantly composed of missense mutations (100% and
92.59%, respectively), especially glycine substitutions. It has
been widely demonstrated that structural mutations have more
severe clinical consequences than quantitative mutations, and
clinical type II-IV are usually caused by mutations altering
structures (1, 4). Similarly, clinical type III was mainly driven
by missense mutations and type I primarily resulted from
quantitative defects in our study, both in de novo and inherited
mutation groups. Repeated mutations are often associated with
CpG dinucleotides, described as mutational hotspots (40). The
mutation of p.Gly337Ser in COL1A2 was detected in four
unrelated families in our study, that is, mutational hot spots.
Four different mutation loci mentioned above were shared in de
novo and inherited mutation groups. Almost all of them had a
consistent clinical type and clinical score between the two
groups, indicating that the severity of the same mutant locus
did not differ between de novo and inherited mutations.

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Mei et al.
Our results revealed that the proportion of type III and
clinical scores were significantly higher in the de novo
mutation group. De novo mutations are genetically different
from inherited mutations and are more damaging because
mutation processes in de novo mutations are happening
between generations without undergoing purifying selection
(41). Zhytnik et al. (14) revealed that in collagen-related OI,
missense mutations and type III occupied a larger proportion in
de novo mutations, and type I was lower compared with inherited
mutations. In our study, the missense mutations in the de novo
COL1A1 mutation group covered a higher proportion than in the
inherited COL1A1 mutation group, though lacking significance,
which may be a reason why there were more type III cases and
higher clinical scores in the de novo mutation group. Although
there was no difference in the mutation spectrum between the de
novo and inherited COL1A2 mutation groups, type III cases and
clinical scores were still higher in de novo mutations than in
inherited COL1A2 mutations, probably due to mutation loci in
de novo mutations with more serious pathogenicity since they
have not undergone purifying selection. Additionally, these
significant results confirmed that our clinical scoring system
could quantitatively reflect the clinical severity of OI. Errors in
DNA replication, exposure to mutagens, or failure to repair DNA
damage can result in de novo mutations (12). Nowadays,
advanced paternal age at conception has been considered the
predominant factor associated with an increasing number of
de novo mutations (9, 11, 42, 43). However, in our study, the
average paternal age at conception was 29.2 years, significantly
younger than the data of the US (29). Thus, paternal age may not
be a critical factor contributing to de novo mutations in our
research. In addition, Wong et al. (44) reported that medically
assisted conception would increase the incidence of de novo
mutations compared with natural conception. However,
recently, a pilot study by Smits et al. (11) showed no impact of
assisted reproductive technologies on the number of de novo
mutations in the genome of the offspring. In our study, the
majority of the pedigrees (98.04%) with de novo mutations were
spontaneous conception, which limited us to investigate the
impact of different methods of conception on de novo mutations.
Mosaicism means an individual develops from a single
fertilized egg but has two or more genetically distinct cell
populations (45). Postzygotic mutations, generated in the first
few cell divisions after fertilization, can result in mosaicism and
exist in many different tissues (43). It has been estimated that
approximately 7% of de novo mutations are early postzygotic
events with high-level mosaicism in the blood (46–48). The
timing of the postzygotic mutations is of vital importance as it
determines the proportion of affected tissues and phenotypes
(45). The recurrence risk is very low, the same as the population
risk, in parents having a child with a postzygotic mutation (45).
Some de novo mutations, but actually because of the parental
germline mosaicism, can be shared by more than one sibling. The
higher the proportion of mosaicism in parental blood cells, the
higher the recurrence risk among siblings (43). Furthermore,
some mutations that arise in the germ cell lineage during early
Frontiers in Endocrinology | www.frontiersin.org
De Novo and Inherited Mutations
embryonic development are undetectable in somatic cells like
blood or buccal mucosa, but can also be passed on to the next
generation (7). Halvorsen et al. (49) revealed that germline
mosaic mutations, which can be transmitted, have not had to
undergo purifying selection as de novo mutations. Pyott et al.
(50) found that the recurrence of lethal OI usually results from
parental mosaicism. Therefore, de novo mutations actually
resulting from paternal germline mosaicism have meaningful
necessities for genetic counseling about recurrence risks. The
detection limit of Sanger sequencing is about 15-20% of somatic
mosaic variants. Therefore, deep sequencing, like SNaPshot
minisequencing assay or whole-exome sequencing, is
recommended to determine the percentage of mosaicism.
Although our comparative study has carried out a meticulous
comparison between de novo and inherited COL1A1/COL1A2
mutations of OI, there are still some limitations to the study
that must be considered: (1) This study was a retrospective review
and suffers from the limitations inherent in such a design. (2) By
adhering strictly to the inclusion criteria, we removed over 36% of
probands in our institution who were confirmed with COL1A1/
COL1A2 pathogenic mutations, which may have resulted in
differences in the prevalence of de novo mutations we report
when compared with other studies, and influence the clinical and
genetical differences in two groups. However, our primary purpose
was to compare the difference between two mutational patterns in
OI, thus we must be 100% confident with the mode of inheritance
of each proband in our study cohort. Therefore, we consider this
as less of a limitation and more of a strength of the study. (3) We
have not adopted deep sequencing in parental samples to make it
clear whether some de novo mutations were actually caused by
parental germinal mosaicism. (4) The research about non-skeletal
phenotypes is not comprehensive in our study, for instance,
cardiovascular abnormalities.
CONCLUSION
Our study showed that the proportion of clinical type III and
clinical scores were significantly higher in de novo mutations
compared with inherited COL1A1 mutations, which may
demonstrate that the pathogenicity of de novo mutations is, on
the whole, more severe than inherited mutations because de novo
variants have not undergone purifying selection. These findings
highlight the further need to investigate the differences between
de novo and inherited mutations in OI and to research the
occurrence of germline mosaicism in the parents of de novo
mutations, which can provide accurate genetic counseling for
both early prenatal and preimplantation diagnosis.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors.
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Mei et al.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by The Ethics Committee of Shanghai Jiao Tong
University Affiliated Sixth People’s Hospital. Written informed
consent to participate in this study was provided by the
participants’ legal guardian/next of kin. Written informed
consent was obtained from the individual(s), and minor(s)’
legal guardian/next of kin, for the publication of any potentially
identifiable images or data included in this article.
AUTHOR CONTRIBUTIONS
ZZ, and HZ designed the research, and revised the manuscript.
YM summarized the clinical data, analyzed the sequencing data,
and drafted the manuscript. HZ contributed to funding
acquisition. All authors contributed to the article and approved
the submitted version.
FUNDING
This research was funded by the National Natural Science
Foundation of China (81870618).
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SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fendo.2022.
935905/full#supplementary-material
Supplementary Figure 1 | De novo and inherited mutation spectrum of
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mutation spectrum of COL1A2. (C) Inherited mutation spectrum of COL1A1. (D)
Inherited mutation spectrum of COL1A2.
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Conflict of Interest: The authors declare that the research was conducted in the
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